Reanalysis of constitutively active rat and human 5-HT7(a) receptors in HEK-293F cells demonstrates lack of silent properties for reported neutral antagonists

Gonzalo Romero, Marta Pujol, Petrus J. Pauwels

The present study reinvestigated a series of 5-HT receptor antagonists at both constitutively active rat and human 5-HT7(a) receptors in HEK-293F cells using the cAMP signalling pathway as a functional read-out. Both rat and human 5-HT7(a) receptors were expressed in similar amounts ([3H]-LSD binding: 1.0 to 1.1 pmol/mg protein). Attenuation of basal cAMP formation by the inverse agonist SB-691673 (1 microM) was slightly larger by the human 5-HT7(a) (-73+/-3 %) than rat 5-HT7(a) receptor (-62+/-3 %). The 5-HT receptor antagonists investigated here displayed systematically inverse agonism. While methiothepin and SB-269970 displayed similar negative intrinsic activity to SB-691673 at the rat 5-HT7(a) receptor, the compounds SB-258719, mesulergine and metergoline displayed some lower negative intrinsic activity (between -38 and -49%). Inverse agonist properties were observed with potencies fitting with their respective binding pIC50 values and pKB values as estimated from antagonist studies with 5-HT. With the exception of SB-258719 and mesulergine, which remained a partial inverse agonist at the human 5-HT7(a) receptor, the other compounds behaved with a similar Emax value to the full inverse agonist SB-691673. In conclusion, none of the 5-HT receptor antagonists investigated displayed silent properties at the rat or human 5-HT7(a) receptor, when these are expressed in a system allowing detection of constitutive activity. They appear to be partial to full inverse agonists, further illustrating that an antagonist is preferentially an inverse agonist when investigated under constitutively active receptor conditions.